APPROACHES TO NUCLEIC ACID EXTRACTION

Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville

Greenville Health System, Greenville, SC hodinka@greenvillemed.sc.edu

Objectives of Talk

Describe the various technologies and advances in nucleic acid purification

Discuss the principles involved in extraction of nucleic acids

Compare and contrast some of the available molecular instruments for nucleic acid extraction

Ingredients Needed for Success with Molecular Assays

Specimens

NA Isolation

Reagents

Facilities

Amplification

Detection

Quality Control

Quality Assurance

Result Analysis &

Interpretation

Specimen Collection & Handling

Specimen collection and handling can have a significant effect on final test results

Integrity of target nucleic acid sequence must be maintained

Clearly defined criteria should be established for proper specimen collection, labeling, preservation, transportation, and storage of specimens

Establish educational programs

Have a firm (but fair) rejection policy

Develop guidelines for specimen processing and carefully manage procedures

Practical Rules of Thumb

As a general rule, submit specimens to laboratory as quickly as possible after collection Keep cold (2-8oC) for short-term transit and storage;

avoid extend times at RT

Freeze to at least -70oC or below for long-term transit or storage following initial processing

Once received by laboratory, processing and storage of specimens for testing should proceed in a timely manner

Store samples in single-use aliquots

Specimen material should not be returned to original container after use for molecular testing

Avoid multiple freeze/thaws; should not exceed three freeze/thaw cycles

Nucleic Acid Extraction

Refers to the release of target nucleic acid from its surrounding material

Key fundamental step to subsequent downstream molecular analysis

Can isolate DNA, RNA or Both

Quality/adequacy of target nucleic acid

Reduction or inactivation of any inhibitory/interfering substances

RNA Extraction Can Be Frustrating

Nucleic Acid Extraction

What’s available? What’s best for me?

Do I use manual or automated systems?

Do I have the needed resources and space?

If automated, which platform should I select?

What number/types of samples will be run?

Do I need DNA, RNA, or both?

What about recovery and quality of purified NA?

What about interfering substances and cross contamination?

How can I best standardize the approach?

Basic Steps of Nucleic Acid Isolation

Pre-processing of samples, if needed

e.g., tissue, stools, whole blood

Lysis of microorganisms to release

nucleic acids

Extraction to remove unwanted cellular

components

Purification of nucleic acids

Bead Beating Cell/Tissue Disruption

Physical disruption before extraction

Grinding Beads

Shaking Homogenizers

MagNA Lyser Cell/Tissue Disruption

Old School-Traditional Methods

Traditional NA Extraction Methods

Treating sample with heat, alkali, sonication

Use of detergents and enzymes

Extraction with organic solvents and precipitation in presence of salts and cold alcohols

Organic Extraction Methods

Accomplished using an organic mixture of phenol-chloroform

Nucleic acid separates into an aqueous phase, lipids in the organic phase, and proteins at the interphase

Keeping the pH at 7.0-8.0 and adding isoamyl alcohol would help keep RNA in aqueous phase

openwetware.org/wiki/phenol/chloroform_extraction

Organic Extraction Methods

Require considerable

labor, time, costs

Multiple

manipulations

Technical burden of

processing multiple

specimens

Difficult to train and

maintain skilled staff

Low Yields

Recovery of partially

degraded material

Co-purification of

contaminants

Variation within and

between extraction

runs

New Wave Technology

Inorganic Extraction Methods

Developed over the years to replace

organic extractions

Samples are lysed

Proteins and other contaminants are

selectively precipitated

The nucleic acids are then precipitated,

washed, and resuspended

Inorganic Extraction Methods

Many commercial kits available

Complete kits for purification of DNA, RNA, or total nucleic acids from biological materials

Selected commercial vendors Epicentre MasterPure

Gentra Systems Puregene

Orca Research IsoQuick

BioMerieux NucliSens

Roche Molecular Isolation Kits

Schleichar & Schuell IsoCode Stix

Mo Bio Molecular Isolation Kits

Advances in NA Isolation

Solid support methods

Significant improvement in speed,

efficiency and standardization of the

process

Increased availability and development

of commercial reagents and systems

Automation

Solid Support Methods

More rapid and user-friendly systems

Use solid matricies (silica or glass

particles) to bind and purify nucleic acids

in presence of chaotropic agents

Many commercial systems based on this

technology

Produce nucleic acid preparations of

high quality and high purity

Spin Column Technology

Lysis Bind Wash Elute

A B C D

Selected Commercial Spin Columns

Qiagen QIAmp, RNeasy

Clontech NucleoSpin

Amresco Cyclo-Prep

Gentra Systems Generation Capture

Stratagene Strata Prep

Roche Molecular High Pure

Thermo Scientific GeneJET

Qiagen Spin Columns

Silica membrane plus chaotropic salts

No organic extraction

No alcohol precipitation

DNA or RNA, Both

Wide range of clinical samples

Mini, midi, and maxi columns

Vacuum manifold for higher specimen numbers and faster processing

Qiagen QIAcube Spin-Column Processing

QIAcube HT 96 Well Platform

Magnetic Particle Technology

Selected Magnetic Particle Kits

Cortex Biochem MegaZorb

Dynal Dynabeads

DNA DIRECT

Roche Molecular DNA Isolation Kit

Magnetic Separators

BioMerieux NucliSens MINI Mag

Semi-automated; 1-12 specimens (10 l-1 ml)

Boom extraction with magnetic silica particles

Total NA from different sample types (10-50 l)

12 extractions in 35 to 40 min (1 instrument)

24 extractions in 45 to 50 min (2 instruments)

Akonni TruTip Technology

< 7 min extraction

For Those Who Desire A Bit More

Need for Automated Systems

Significant portion of total testing time and effort is dedicated to sample preparation

Simplification of specimen processing

Need for uniformity and consistency

Direct cost savings

Staffing problems

The Modern Family of Instruments

Automated Sample Processing

Many options are now available Different levels of automation (semi to fully

automated)

Large and small platforms; different sizes, shapes, weights

Varied batch sizes depending on system

Sample processing capacity (1-96 per run)

Various sample input volumes

Variety of chemistries for purification of DNA, RNA or total nucleic acids

Some have post elution protocols/functions

Automated NA Isolation Systems

bioMerieux: mini Mag & easy Mag Extractors

Roche Molecular: MagNA Pure LC, Compact, & 96; AmpliPrep

Qiagen: QIAcube, EZ1 Advanced, EZ1 Advanced XL, QIAsymphony SP

Abbott Molecular: m2000sp Sample Prep Station

Beckman Coulter:

SPRI-TE

NorDiag: Arrow

Promega: Maxwell-16

Invitrogen: iPrep

BioChain: AnaPrep 12

Autogene: QuickGene

610L, 810, 810 Mini

Selected Vendors of Semi and Fully Automated Systems

Compact Models

Compact Systems

Roche MagNA Pure Compact QIAGEN BioRobot EZ1 & EZ1 Advanced/Advanced XL

Beckman Coulter SPRI-TE NorDiag Arrow Promega Maxwell-16

Luxury Sedans

Bugatti 16C Galibier

Larger Platforms

BioMerieux NucliSens EASYMAG

Roche MagNA Pure LC 2.0

Roche MagNA Pure 96

Roche AmpliPrep

QIAGEN QIAsymphony SP

Hologic Gen-Probe Panther

Abbott Molecular m2000

Advantages of Automation

Easy/practical to use

Some truly walk away

Reduces hands-on time, sample manipulation

Decreases labor costs, increases throughput

Process variety of sample types and volumes

Standardized technology

Simplified software

Minimize chance of

cross contamination

UV light

decontamination

Minimal maintenance

and down time

Post elution functions

for some

Disadvantages of Automation

Which instrument(s) to choose

Expense

Varied batch sizes depending on system

Varied kit availability for DNA, RNA, total NA

Some samples may require external processing prior to system extraction

Some instruments require manual additions of reagents during extraction

No or slow post elution protocols

Size, shape, and weight

NA Yield, Integrity, Purity

Concentration of DNA 1 A260 U dsDNA = 50 g/ml

1 A260 U ssDNA = 33 g/ml

Purity of DNA Pure DNA: A260/A280 1.8

An A260/A280 < 1.8 (Contaminated with proteins and aromatic reagents)

An A260/A280 > 2.0 RNA contamination

Concentration of RNA

1 A260 U ssRNA = 40 g/ml

Purity of RNA

Pure RNA: A260/A280 2.0

An A260/A280 < 2.0

(Contaminated with

proteins and aromatic

reagents)

Analyzing DNA Analyzing RNA

Storage of Purified Nucleic Acids

Ideally, purified nucleic acids should be used immediately for downstream amplification and detection

If delayed, store under appropriate conditions until testing can be completed

DNA is more stable than RNA and can be stored for long periods at even 4oC in a suitable TRIS buffer

Being more labile, RNA should be stored at -70oC or colder in the same buffer as DNA

As a general rule in my laboratory, storage of all purified nucleic acid preparations are done at

-70oC or colder.

It’s All in the Extraction